Mutation of the Sp1 binding site in the 5′ flanking region of SRY causes sex reversal in rabbits

// Yuning Song 1,* , Tingjun Liu 1,* , Yong Wang 1 , Jichao Deng 1 , Mao Chen 1 , Lin Yuan 1 , Yi Lu 1 , Yuxin Xu 1 , Haobin Yao 1 , Zhanjun Li 1 and Liangxue Lai 1,2 1 Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, Changchun, China 2 Key Laboratory of Regenerative Biology, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China * These authors have contributed equally to this work Correspondence to: Zhanjun Li, email: // Liangxue Lai, email: // Keywords : Sp1; SRY; CRISPR/Cas9; sex reversed; Pathology Section Received : January 25, 2017 Accepted : March 28, 2017 Published : April 09, 2017 Abstract Sex-determining region Y is a crucial gene that initiates male sex determination in mammals. Mutations of the Sp1-binding site in the 5’ flanking region of SRY are associated with clinical male-to-female sex reversal syndrome, although such occurrences are rare and, until now, have not been reported in animal models. In this study, we mutated Sp1-binding sites in the 5’ flanking region of the rabbit SRY gene using the CRISPR/Cas9 system. As expected, the SRY -Sp1 knockout rabbits had female external and internal genitalia and exhibited normal female copulatory behaviors, but they were infertile, and the adults displayed reduced follicles. Interestingly, we successfully obtained offspring from sex-reversed SRY -Sp1 knockout rabbits using embryo transfer. In summary, our study demonstrates that Sp1 is a major regulator in SRY gene transcription, and mutations of the Sp1 binding sites (Sp1-B and Sp1-C) in the 5’ flanking region of SRY induce sex reversal in rabbits, which can be used as targets for clinical research of male-to-female sex reversal syndrome. Additionally, we provide the first evidence that sex reversal syndrome patients have the potential to become pregnant with the use of embryo transfer.